The Epithelial Na+ Channel (ENaC) is the rate limiting step for Na+ absorption across many epithelia. In the airways of cystic fibrosis (CF) patients, ENaC is abnormally hyperactive leading to disproportionate Na+ absorption and a depletion of airway surface liquid (ASL) volume. ENaC hyperactivity contributes to mucus stasis and increased incidence of airway infections that frequently lead to the death of the patient. Despite previous efforts, there are no existing therapies to treat abnormal ENaC activity in the lung. Using a proteomic screen, we have identified the protein SPLUNC1 as a potent allosteric inhibitor of ENaC that binds extra-cellularly to ENaC, reducing the amount of ENaC in the plasma membrane and subsequently limiting ENaC activity. We have also identified the ENaC-inhibitory domain of SPLUNC1, and have synthesized small peptide that corresponds to this domain, called SPX-101. This peptide robustly inhibits ENaC and prevents ASL hyperabsorption in CF airway cultures for 24 h following a single dose. SPX-101 continues to function in the presence of neutrophil elastase, which is highly abundant in CF airways, suggesting that this peptide may be therapeutically beneficial in the treatment of CF lung disease. We have also demonstrated that SPX-101 improves survival in a transgenic mouse model of CF that specifically overexpresses ENaC in the lung epithelia (?ENaC-Tg) and that SPX-101 can be effectively delivered to the lung via nebulization. Notably, SPX-101 does not induce diuresis or hyperkalemia when delivered by intravenous infusion in rats. Furthermore, nebulized SPX-101 remains sequestered within the lung and <1% of the dose administered to the lung reaches systemic circulation in rats. To advance the development of a SPX-101 for the treatment of CF we are seeking this Phase I SBIR grant to: (i) measure the effect of SPX-101 in rat, canine and ovine bronchial epithelial cells to demonstrate the relevance of these species for toxicological and translational studies, and (ii) define the maximal effective dose and fully power the dose dependent effects of SPX-101 in a CFTR inhibitor- induced inhibition of tracheal mucus velocity in sheep. Completion of the proposed work will lead to a Phase II application that will be aimed at completion of toxicology studies and human clinical trials of this novel therapy for CF.